Dual-pore device

ABSTRACT

Provided is a device comprising an upper chamber, a middle chamber and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore, wherein the first pore and second pore are about 1 nm to about 100 nm in diameter, and are about 10 nm to about 1000 nm apart from each other, and wherein each of the chambers comprises an electrode for connecting to a power supply. Methods of using the device are also provided, in particular for sequencing a polynucleotide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 USC 111(a) whichclaims the benefit of and priority to pending U.S. application Ser. No.13/882,191 titled “Dual-pore device” filed 28-Apr.-2013; which itselfclaims the benefit of and priority to PCT/US12/47107 filed 18 Jul. 2012titled “Dual-pore device”; which itself claims the benefit of andpriority to U.S. provisional application Ser. No. 61/572,843 filed Jul.20, 2011 titled “Dual-nanopore electronics configuration to co-trapindividual DNA molecules for sequencing and single molecule science”.The content of all above applications is incorporated by reference inits entirety into the present disclosure.

BACKGROUND

A nanopore is a nano-scale opening that forms naturally as a proteinchannel in a lipid membrane (a biological pore), or is engineered bydrilling or etching the opening in a solid-state substrate (asolid-state pore). When such a nanopore is incorporated into ananodevice comprising two chambers which are separated by the nanopore,a sensitive patch-clamp amplifier can be used to apply a trans-membranevoltage and measure ionic current through the pore.

Nanopores offer great promise for inexpensive whole genome DNAsequencing. In this respect, individual DNA molecules can be capturedand driven through the pore by electrophoresis, with each capture eventdetected as a temporary shift in the ionic current. The sequence of aDNA molecule can then be inferred from patterns within the shifted ioniccurrent record, or from some other auxiliary sensor in or near thenanopore, as DNA passes through the pore channel.

In principle, a nanopore sequencer can eliminate the needs for sampleamplification, the use of enzymes and reagents used for catalyticfunction during the sequencing operation, and optics for detection ofsequencing progress, some or all of which are required by theconventional sequencing-by-synthesis methods.

Nanopore sensors are purely electrical, and can detect DNA inconcentrations/volumes that are no greater than what is available from ablood or saliva sample. Additionally, nanopores promise to dramaticallyincrease the read-length of sequenced DNA, from 450 bases to greaterthan 10,000 bases.

There are two principle obstacles to nanopore sequencing: (1) the lackof sensitivity sufficient to accurately determine the identity of eachnucleotide in a nucleic acid for de novo sequencing (the lack ofsingle-nucleotide sensitivity), and (2) the ability to regulate thedelivery rate of each nucleotide unit through the nanopore duringsensing. While many research groups are developing and improvingnanopores to address obstacle 1, there is no method for addressingobstacle 2 that does not involve the use of enzymes or optics, both ofwhich work only in specialized nanopore techniques and which incurhigher complexity and cost compared to purely electrical methods.

SUMMARY

In one embodiment, provided is a device comprising an upper chamber, amiddle chamber and a lower chamber, wherein the upper chamber is incommunication with the middle chamber through a first pore, and themiddle chamber is in communication with the lower chamber through asecond pore, wherein the first pore and second pore are about 1 nm toabout 100 nm in diameter, and are about 10 nm to about 1000 nm apartfrom each other, and wherein each of the chambers comprises an electrodefor connecting to a power supply.

In one aspect, the first and second pores are substantially coaxial.

In one aspect, the device comprises a material selected from the groupconsisting of silicon, silicon nitride, silicon dioxide, graphene,carbon nanotubes, TiO₂, HfO₂, Al₂O₃, metallic layers, glass, biologicalnanopores, membranes with biological pore insert, and combinationsthereof.

In one aspect, the first pore and the second pore are about 0.3 nm toabout 100 nm in depth.

In one aspect, the power supply is configured to provide a first voltagebetween the upper chamber and the middle chamber, and a second voltagebetween the middle chamber and the lower chamber, and wherein the firstvoltage and the second voltage are independently adjustable.

In one aspect, the power supply comprises a voltage-clamp system or apatch-clamp system to generate each of the first and second voltages. Inone aspect, the middle chamber is adjusted to be ground relative to thetwo voltages. In one aspect, the middle chamber comprises a medium forproviding conductance between each of the pores and the electrode in themiddle chamber.

In some aspects, the power supply, such as the voltage-clamp system orthe patch-clamp system, is further configured to measure the ioniccurrent through each of the pores.

Another embodiment provides a device comprising an upper chamber, amiddle chamber and a lower chamber, wherein the upper chamber is incommunication with the middle chamber through a first pore, and themiddle chamber is in communication with the lower chamber through asecond pore, and wherein the first pore and second pore are about 1 nmto about 100 nm in diameter, and are about 10 nm to about 1000 nm apartfrom each other; and an electrode in each of the chambers for connectingto a voltage-clamp or patch-clamp system to apply a voltage across andmeasuring ionic current through each of the pores, wherein the electrodein the middle chamber is connected to a common ground of the twovoltage-clamp or patch-clamp systems.

Also provided, in one embodiment, is a method for controlling themovement of a charged polymer through a pore, comprising: (a) loading asample comprising a charged polymer in one of the upper chamber, middlechamber or lower chamber of the device of any of the above embodiments,wherein the device is connected to a voltage-clamp or patch-clamp systemfor providing a first voltage between the upper chamber and the middlechamber, and a second voltage between the middle chamber and the lowerchamber; (b) setting an initial first voltage and an initial secondvoltage so that the polymer moves between the chambers, thereby locatingthe polymer across both the first and second pores; and (c) adjustingthe first voltage and the second voltage so that both voltages generateforce to pull the charged polymer away from the middle chamber, whereinthe two voltages are different in magnitude, under controlledconditions, so that the charged polymer moves across both pores in onedirection and in a controlled manner.

In one aspect, the controlled manner of delivery is established byactive control or feedback control of the first or second or bothvoltages, with either or both as a feedback function of the first orsecond or both ionic current measurements. A non-limiting exampleincludes keeping the second voltage constant, and using the second ioniccurrent as feedback for feedback or active control of the first voltage,to established controlled delivery of a charged polymer in eitherdirection. Accordingly, in one aspect, the first voltage is adjustedbased on a measured ionic current across the second pore.

In one aspect, the sample is loaded into the upper chamber and theinitial first voltage is set to pull the charged polymer from the upperchamber to the middle chamber and the initial second voltage is set topull the polymer from the middle chamber to the lower chamber.

In another aspect, the sample is loaded into the middle chamber and theinitial first voltage is set to pull the charged polymer from the middlechamber to the upper chamber and the initial second voltage is set topull the charged polymer from the middle chamber to the lower chamber.

In one aspect, the charged polymer is a polynucleotide or a polypeptide.In one aspect, the charged polymer is a polynucleotide such as, but notlimited to, a double-stranded DNA, single-stranded DNA, double-strandedRNA, single-stranded RNA, or DNA-RNA hybrid.

In one aspect, the adjusted first voltage and second voltage at step (c)are about 10 times to about 10,000 times as high, in magnitude, as thedifference between the two voltages.

In one aspect, the method further comprises identifying a monomer unitof the polymer by measuring an ionic current across one of the poreswhen the monomer unit passes that pore. In one aspect, the monomer unitis a nucleotide. In another aspect, the monomer unit is a nucleotidepair. Single nucleotides and nucleotide pairs, in some aspects, can bedetected in one molecule. For instance, such a molecule can have aduplex segment in a longer and otherwise single-stranded polynucleotide,with the duplex formed partially or fully by Watson-Crick complementarybase pairing.

In one aspect, the monomer is bound to a molecule, such as a DNA-bindingprotein, or a nano-particle. Non-limiting examples of DNA-bindingproteins include RecA and sequence-specific DNA-binding protein such asphage lambda repressor, NF-κB and p53. Non-limiting examples ofnano-particles include quantum dots and fluorescent labels.

In one aspect, the polymer is attached to a solid support, such as abead, at one end of the polymer.

Yet another embodiment provides a method for determining the sequence ofa polynucleotide, comprising: (a) loading a sample comprising apolynucleotide in the upper chamber of the device of any of the aboveembodiments, wherein the device is connected to a voltage-clamp orpatch-clamp system for providing a first voltage between the upperchamber and the middle chamber, and a second voltage between the middlechamber and the lower chamber, wherein the polynucleotide is optionallyattached to a solid support at one end of the polynucleotide; (b)setting an initial first voltage and an initial second voltage so thatthe polynucleotide moves from the upper chamber to the middle chamberand from the middle chamber to the lower chamber, thereby locating thepolymer across both the first and second pores; (c) adjusting the firstvoltage and the second voltage so that both voltages generate force topull the polynucleotide away from the middle chamber, wherein the twovoltages are different in magnitude, under controlled conditions, sothat the polynucleotide moves across both pores in one direction and ina controlled manner; and (d) identifying each nucleotide of thepolynucleotide that passes through one of the pores, by measuring anionic current across the pore when the nucleotide passes that pore.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures of the accompanying drawings describe provided embodimentsby way of illustration only, in which:

FIG. 1(I)-(III) illustrate a two-pore (dual-pore) device. (I) Schematicof dual-pore chip and dual-amplifier electronics configuration forindependent voltage control (V₁, V₂) and current measurement (I₁, I₂) ofeach pore. Chambers (A-C) are volumetrically separated except by commonpores. Feasible chip parameters are an inter-pore distance 10-500 nm,membrane thickness 0.3-50 nm, and pore diameters 1-100 nm. (II)Electrically, V₁ and V₂ are principally across each nanopore resistance,by constructing a device that minimizes all access resistances toeffectively decouple I₁ and I₂. (III) Competing voltages will be usedfor control, with blue arrows showing the direction of each voltageforce. Assuming pores with identical voltage-force influence and using|V₁|=|V₂|+δV, the value δV>0 (<0) is adjusted for tunable motion in theV₁ (V₂) direction. In practice, although the voltage-induced force ateach pore will not be identical with V₁=V₂, calibration experiments canidentify the required voltage bias that will result in equal pullingforces, for a given two-pore chip, and variations around that bias canthen be used for directional control; and

FIG. 2 shows an external view of a two-pore housing device having aChamber A, Chamber B and Chamber C, each having an opening for fluidicaccess and sample loading. A dual-pore chip is placed between twogaskets, with each gasket part of each portion of the housing device,and the two portions rotate around a hinge (middle top) to open andclose the housing around the chip.

Some or all of the figures are schematic representations forexemplification; hence, they do not necessarily depict the actualrelative sizes or locations of the elements shown. The figures arepresented for the purpose of illustrating one or more embodiments withthe explicit understanding that they will not be used to limit the scopeor the meaning of the claims that follow below.

DETAILED DESCRIPTION

Throughout this application, the text refers to various embodiments ofthe present nutrients, compositions, and methods. The variousembodiments described are meant to provide a variety of illustrativeexamples and should not be construed as descriptions of alternativespecies. Rather it should be noted that the descriptions of variousembodiments provided herein may be of overlapping scope. The embodimentsdiscussed herein are merely illustrative and are not meant to limit thescope of the present invention.

Also throughout this disclosure, various publications, patents andpublished patent specifications are referenced by an identifyingcitation. The disclosures of these publications, patents and publishedpatent specifications are hereby incorporated by reference into thepresent disclosure to more fully describe the state of the art to whichthis invention pertains.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “an electrode” includes a plurality ofelectrodes, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thedevices and methods include the recited components or steps, but notexcluding others. “Consisting essentially of” when used to definedevices and methods, shall mean excluding other components or steps ofany essential significance to the combination. “Consisting of” shallmean excluding other components or steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

All numerical designations, e.g., distance, size, temperature, time,voltage and concentration, including ranges, are approximations whichare varied (+) or (−) by increments of 0.1. It is to be understood,although not always explicitly stated that all numerical designationsare preceded by the term “about”. It also is to be understood, althoughnot always explicitly stated, that the components described herein aremerely exemplary and that equivalents of such are known in the art.

Two-Pore Device

One embodiment of the present disclosure provides a two-pore device. Thedevice includes three chambers and two pores that enable fluidcommunication between the chambers. Further, each of the chamberscontains an electrode for connecting to a power supply so that aseparate voltage can be established across each of the pores between thechambers.

In accordance with one embodiment of the present disclosure, provided isa device comprising an upper chamber, a middle chamber and a lowerchamber, wherein the upper chamber is in communication with the middlechamber through a first pore, and the middle chamber is in communicationwith the lower chamber through a second pore, wherein the first pore andsecond pore are about 1 nm to about 100 nm in diameter, and are about 10nm to about 1000 nm apart from each other, and wherein each of thechambers comprises an electrode for connecting to a power supply.

With reference to FIG. 1(I), the device includes an upper chamber(Chamber A), a middle chamber (Chamber B), and a lower chamber (ChamberC). The chambers are separated by two separating layers or membranes(101 and 102) each having a separate pore (111 and 112). Further, eachchamber contains an electrode (121, 122 and 123) for connecting to apower supply. It is apparent that the annotation of upper, middle andlower chamber is in relative terms and does not indicate that, forinstance, the upper chamber is placed above the middle or lower chamberrelative to the ground, or vice versa.

Each of the pores (111 and 112) independently has a size that allows asmall or large molecule or microorganism to pass. In one aspect, eachpore is at least about 1 nm in diameter. Alternatively, each pore is atleast about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100nm in diameter.

In one aspect, the pore is no more than about 100 nm in diameter.Alternatively, the pore is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 or 10 nm in diameter.

In one aspect, the pore has a diameter that is between about 1 nm andabout 100 nm, or alternatively between about 2 nm and about 80 nm, orbetween about 3 nm and about 70 nm, or between about 4 nm and about 60nm, or between about 5 nm and about 50 nm, or between about 10 nm andabout 40 nm, or between about 15 nm and about 30 nm.

In some aspects, the pore has a substantially round shape.“Substantially round”, as used here, refers to a shape that is at leastabout 80 or 90% in the form of a cylinder. In some embodiments, the poreis square, rectangular, triangular, oval, or hexangular in shape.

Each of the pores (111 and 112) independently has a depth. In oneaspect, each pore has a depth that is least about 0.3 nm. Alternatively,each pore has a depth that is at least about 0.6 nm, 1 nm, 2 nm, 3 nm, 4nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm.

In one aspect, each pore has a depth that is no more than about 100 nm.Alternatively, the depth is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 or 10 nm.

In one aspect, the pore has a depth that is between about 1 nm and about100 nm, or alternatively between about 2 nm and about 80 nm, or betweenabout 3 nm and about 70 nm, or between about 4 nm and about 60 nm, orbetween about 5 nm and about 50 nm, or between about 10 nm and about 40nm, or between about 15 nm and about 30 nm.

In one aspect, the pores are spaced apart at a distance that is betweenabout 10 nm and about 1000 nm. In one aspect, the distance is at leastabout 10 nm, or alternatively at least about 20 nm, 30 nm, 40 nm, 50 nm,60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm.In another aspect, the distance is no more than about 1000 nm, 900 nm,800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 150 nm,or 100 nm. In yet another aspect, the distance is between about 20 nmand about 800 nm, between about 30 nm and about 700 nm, between about 40nm and about 500 nm, or between about 50 nm and about 300 nm.

The two pores can be arranged in any position so long as they allowfluid communication between the chambers and have the prescribed sizeand distance between them. In one aspect, the pores are placed so thatthere is no blockage directly between them. Still, in one aspect, thepores are substantially coaxial, as illustrated in FIG. 1(I).

In one aspect, the device, through the electrodes in the chambers, isconnected to one or more power supply. In some aspects, the power supplyis comprised of a voltage-clamp or a patch-clamp for supplying thevoltage across each pore, which can also measure the current througheach pore independently. In this respect, the power supply can set themiddle chamber to a common ground for both voltage sources. In oneaspect, the power supply is configured to provide a first voltagebetween the upper chamber (e.g., Chamber A in FIG. 1(I)) and the middlechamber (e.g., Chamber B in FIG. 1(I)), and a second voltage between themiddle chamber and the lower chamber (e.g., Chamber C in FIG. 1(I)).

In some aspects, the first voltage and the second voltage areindependently adjustable. In one aspect, the middle chamber is adjustedto be ground relative to the two voltages (illustrated in FIG.1(I-III)). In one aspect, the middle chamber comprises a medium forproviding conductance between each of the pores and the electrode in themiddle chamber. In one aspect, the middle chamber comprises a medium forproviding a resistance between each of the pores and the electrode inthe middle chamber. Keeping such a resistance sufficiently small,relative to the nanopore resistances, is useful for decoupling the twovoltages and currents across the pores, which is helpful for theindependent adjustment of the voltages.

Adjustment of the voltages can be used to control the movement ofcharged particles in the chambers. For instance, when both voltages areset in the same direction, a properly charged particle can be moved fromthe upper chamber to the middle chamber and to the lower chamber, or theother way around, sequentially. Otherwise, a charged particle can bemoved from either the upper or the lower chamber to the middle chamberand kept there.

The adjustment of the voltages in the device can be particularly usefulfor controlling the movement of a large molecule, such as a chargedpolymer, that is long enough to cross both of the pores at the sametime. In such an aspect, the movement and the rate of movement of themolecule can be controlled by the relative magnitude and direction ofthe voltages, which will be further described below.

The device can contain materials suitable for holding liquid samples, inparticular, biological samples, and/or materials suitable fornanofabrication. In one aspect, such materials include dielectricmaterials such as, but not limited to, silicon, silicon nitride, silicondioxide, graphene, carbon nanotubes, TiO₂, HfO₂, Al₂O₃, or othermetallic layers, or any combination of these materials. A single sheetof graphene forms a membrane ˜0.3 nm thick, and can be used as thepore-bearing membrane, for example.

Devices that are microfluidic and that house two-pore microfluidic chipimplementations can be made by a variety of means and methods. For amicrofluidic chip comprised of two parallel membranes, both membranescan be simultaneously drilled by a single beam to form two concentricpores, though using different beams on each side of the membranes isalso possible in concert with any suitable alignment technique. Ingeneral terms, the housing ensures sealed separation of Chambers A-C. Inone aspect, the housing would provide minimal access resistance betweenthe voltage electrodes (two sources and one ground) and the nanopores,to ensure that each voltage is applied principally across each pore.

In one aspect, FIG. 2 shows an external view of another embodiment ofthe device. In FIG. 2, the device contains a microfluidic chip (labeledas “Dual-core chip”) comprised of two parallel membranes connect byspacers. Each membrane contains a pore (not shown) drilled by a singlebeam through the center of the membrane. Further, the device preferablyhas a Teflon® housing for the chip. The housing ensures sealedseparation of Chambers A-C and provides minimal access resistance forthe electrolyte to ensure that each voltage is applied principallyacross each pore.

More specifically, the pore-bearing membranes can be made with TEM(transmission electron microscopy) grids with 5-100 nm thick silicon,silicon nitride, or silicon dioxide windows. Spacers can be used toseparate the membrane, using an insulator (SU-8, photoresist, PECVDoxide, ALD oxide, ALD alumina) or an evaporated metal (Ag, Au, Pt)material, and occupying a small volume within the otherwise aqueousportion of Chamber B between the membranes. The holder is seated in anaqueous bath that comprises io the largest volumetric fraction ofChamber B. Chambers A and C are accessible by larger diameter channels(for low access resistance) that lead to the membrane seals.

A focused electron or ion beam can be used to drill pores through themembranes, naturally aligning them. The pores can also be sculpted(shrunk) to smaller sizes by applying the correct beam focus to eachlayer. Any single nanopore drilling method can also be used to drill thepair of pores in the two membranes, with consideration to the drilldepth possible for a given method and the thickness of the membranes.Predrilling a micro-pore to a prescribed depth and then a nanoporethrough the remainder of the membranes is also possible, to furtherrefine membrane thicknesses.

In another aspect, insertion of biological nanopores into solid-statenanopores to form a hybrid pore can be used in either or both nanoporesin the two-pore method (Hall et al., Nat. Nanotech., 5(12):874-7, 2010).The biological pore can increase the sensitivity of the ionic currentmeasurements, and are useful when only single-stranded polynucleotidesare to be captured and controlled in the two-pore device, e.g., forsequencing.

Controlling Movement of Molecules with a Two-Pore Device

By virtue of the voltages present at the pores of the device, chargedmolecules can be moved through the pores between chambers. Speed anddirection of the movement can be controlled by the magnitude anddirection of the voltages. Further, because each of the two voltages canbe independently adjusted, the movement and speed of a charged moleculecan be finely controlled in each chamber.

For instance, the device can be used to admix two positively chargedmolecules in a controlled manner. To this end, the first molecule isinitially loaded in the upper chamber and the second in the lowerchamber. A first voltage across the first port can induce movement ofthe first molecule into the middle chamber from the upper chamber.Likewise, a second voltage, in the opposite direction to the firstvoltage, can induce movement of the second molecule into the middlechamber from the lower chamber. Due to the opposite directions of thevoltages, both molecules will be kept in the middle chamber so as toreact with each other. Further, by adjusting the relative magnitudes ofthe voltages, the inflow speeds of each molecules can be fine tuned,leading to controlled reaction.

Another example concerns a charged polymer, such as a polynucleotide,having a length that is longer than the combined distance that includesthe depth of both pores plus the distance between the two pores. Forexample, a 1000 bp dsDNA is ˜340 nm in length, and would besubstantially longer than the 40 nm spanned by two 10 nm-length poresseparated by 20 nm. In a first step, the polynucleotide is loaded intoeither the upper or the lower chamber. By virtue of its negative chargeunder a physiological condition (˜pH 7.4), the polynucleotide can bemoved across a pore on which a voltage is applied. Therefore, in asecond step, two voltages, in the same direction and at the same orsimilar magnitudes, are applied to the pores to induce movement of thepolynucleotide across both pores sequentially.

At about time when the polynucleotide reaches the second pore, one orboth of the voltages can be changed. Since the polynucleotide is longerthan the distance covering both pores, when the polynucleotide reachesthe second pore, it is also in the first pore. A prompt change ofdirection of the voltage at the first pore, therefore, will generate aforce that pulls the polynucleotide away from the second pore(illustration in FIG. 1(III)).

If, at this point, the magnitude of the voltage-induced force at thefirst pore is less than that of the voltage-induced force at the secondpore, then the polynucleotide will continue crossing both pores towardsthe second pore, but at a lower speed. In this respect, it is readilyappreciated that the speed and direction of the movement of thepolynucleotide can be controlled by the directions and magnitudes ofboth voltages. As will be further described below, such a fine controlof movement has broad applications.

Accordingly, in one aspect, provided is a method for controlling themovement of a charged polymer through a pore. The method entails (a)loading a sample comprising a charged polymer in one of the upperchamber, middle chamber or lower chamber of the device of any of theabove embodiments, wherein the device is connected to a power supply forproviding a first voltage between the upper chamber and the middlechamber, and a second voltage between the middle chamber and the lowerchamber; (b) setting an initial first voltage and an initial secondvoltage so that the polymer moves between the chambers, thereby locatingthe polymer across both the first and second pores; and (c) adjustingthe first voltage and the second voltage so that both voltages generateforce to pull the charged polymer away from the middle chamber(voltage-competition mode), wherein the two voltages are different inmagnitude, under controlled conditions, so that the charged polymermoves across both pores in either direction and in a controlled manner.

For the purpose of establishing the voltage-competition mode in step(c), the relative force exerted by each voltage at each pore is to bedetermined for each two-pore device used, and this can be done withcalibration experiments by observing the influence of different voltagevalues on the motion of the polynucleotide (motion can be measured bysensing location-known and detectable features in the polynucleotide,with examples of such features detailed later in this provisionaldocument). If the forces are equivalent at each common voltage, forexample, then using the same voltage value at each pore (with commonpolarity in upper and lower chambers relative to grounded middlechamber) creates a zero net motion in the absence of thermal agitation(the presence and influence of Brownian motion is discussed below). Ifthe forces are not equivalent at each common voltage, then achievingequal forcing requires identification and use of a larger voltage at thepore that experiences a weaker force at the common voltage. Calibrationfor voltage-competition mode is required for each two-pore device, andwould be required for specific charged polymers or molecules for whichfeatures that pass through each pore influence the force.

In one aspect, the sample is loaded into the upper chamber and theinitial first voltage is set to pull the charged polymer from the upperchamber to the middle chamber and the initial second voltage is set topull the polymer from the middle chamber to the lower chamber. Likewise,the sample can be initially loaded into the lower chamber.

In another aspect, the sample is loaded into the middle chamber and theinitial first voltage is set to pull the charged polymer from the middlechamber to the upper chamber and the initial second voltage is set topull the charged polymer from the middle chamber to the lower chamber.

In some aspects, the charged polymer is a polynucleotide or apolypeptide. In a particular aspect, the charged polymer is apolynucleotide. Non-limiting examples of polynucleotides includedouble-stranded DNA, single-stranded DNA, double-stranded RNA,single-stranded RNA, and DNA-RNA hybrids.

In one aspect, the adjusted first voltage and second voltage at step (c)are about 10 times to about 10,000 times as high, in magnitude, as thedifference between the two voltages. For instance, the two voltages are90 mV and 100 mV, respectively. The magnitude of the voltages (˜100 mV)is about 10 times of the difference between them, 10 mV. In someaspects, the magnitude of the voltages is at least about 15 times, 20times, 25 times, 30 times, 35 times, 40 times, 50 times, 100 times, 150times, 200 times, 250 times, 300 times, 400 times, 500 times, 1000times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000times, 8000 times or 9000 times as high as the difference between them.In some aspects, the magnitude of the voltages is no more than about10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times,4000 times, 3000 times, 2000 times, 1000 times, 500 times, 400 times,300 times, 200 times, or 100 times as high as the difference betweenthem.

In one aspect, real-time or on-line adjustments to first voltage andsecond voltage at step (c) are performed by active control or feedbackcontrol using dedicated hardware and software, at clock rates up tohundreds of megahertz. Automated control of the first or second or bothvoltages is based on feedback of the first or second or both ioniccurrent measurements.

Analysis of Molecules with a Two-Pore Device

The two-pore device of the present disclosure can be used to carry ouranalysis of molecules or particles that move or are kept within thedevice by virtue of the controlled voltages applied over the pores. Inone aspect, the analysis is carried out at either or both of the pores.Each voltage-clamp or patch-clamp system measures the ionic currentthrough each pore, and this measured current is used to detect thepresence of passing charged particle or molecules, or any featuresassociated with a passing charged particle or molecule.

As provided above, a polynucleotide can be loaded into both pores by twovoltages having the same direction. In this example, once the directionof the voltage applied at the first pore is inversed and the newvoltage-induced force is slightly less, in magnitude, than thevoltage-induced force applied at the second pore, the polynucleotidewill continue moving in the same direction, but at a markedly lowerspeed. In this respect, the amplifier supplying voltage across thesecond pore also measures current passing through the second pore, andthe ionic current then determines the identification of a nucleotidethat is passing through the pore, as the passing of each differentnucleotide would give rise to a different current signature (e.g., basedon shifts in the ionic current amplitude). Identification of eachnucleotide in the polynucleotide, accordingly, reveals the sequence ofthe polynucleotide.

In some aspects, repeated controlled delivery for re-sequencing apolynucleotide further improves the quality of sequencing. Each voltageis alternated as being larger, for controlled delivery in eachdirection. Also contemplated is that the two currents through the twopores can be correlated to improved accuracy. It is contemplated thatBrownian motion may cause fluctuations in the motion of a molecule,affecting controlled delivery of the molecule. Such an effect, however,can be minimized or avoided by, e.g., during DNA sequencing, repeatedcontrolled delivery of the DNA and averaging the sequencingmeasurements. Still further, it is contemplated that the impact ofBrownian motion on the controlled motion of large molecules, such aspolynucleotides and polypeptides, would be insignificant in particularwhen competing forces are pulling the larges molecules apart, generatingtension within the molecule.

Such a method provides a ready solution to problems that have not beensolved in the prior art.

For instance, it is known that there are two competing obstacles toachieve the controlled delivery and accurate sensing required fornanopore sequencing. One is that a relatively high voltage is required,at the pore, to provide enough sequencing sensitivity. On the otherhand, high voltages lead to fast passing of a polynucleotide through thepore, not allowing sufficient time for identification of eachnucleotide.

More specifically, the nanopore sequencing platform requires that therate of polynucleotide passage through the pore be controlled to 1ms/nucleotide (nt) or slower, while still generating asequence-sensitive current. This requires sufficiently highsignal-to-noise for detecting current signatures (high voltage isbetter), but sufficiently slow motion of the molecule through the poreto ensure measurements are within the recording bandwidth (low voltageis better). In single pore implementations, polynucleotide speed isproportional to voltage, so higher voltage is better for sensing butworse for reducing polynucleotide speed: rates are 1 μs/nt and faster(>1000 times too fast) at voltages that promote polynucleotide capture.On the other hand, lower voltages reduce sensing performance, and alsoincrease the relative contribution of rate fluctuations caused byBrownian motion that will undermine read accuracy.

Other than what is described herein, there are currently no methods foraddressing these obstacles that do not involve the use of enzymes oroptics, both of which work only in specialized nanopore techniques.

Several approaches have been proposed to address the problem associatedwith the lack of sensing capability, and under low voltages. One is toengineer biological nanopores to improve their sensitivity. Another isto use graphene membranes, which as a single sheet are thinner than thedistance between nucleotides in ssDNA. Still another is the use of anauxiliary current measured in close proximity to the nanopore (e.g.,tunneling currents).

Biological nanopores have been tested in the first approach. Theα-hemolysin nanopore is the most commonly used biological pore inresearch. Studies have shown that α-hemolysin can resolve singlenucleotide substitutions in homopolymers and abasic (1′,2′-dideoxy)residues within otherwise all-nucleobase DNA. However, single nucleotidesensitivity is not possible in heteromeric DNA with wild-type (WT)α-hemolysin, for which the ionic current is influenced by ˜10nucleotides in the channel. Protein engineering of α-hemolysin has beenused to improve its sensitivity for DNA analysis and sequencing. Onesuch mutant pore uses α-hemolysin with a covalently attached molecularadapter (Clarke et al., Nat. Nanotech, 4(4):265-70, 2009) that iscapable of discriminating the four nucleoside 5′-monophosphate moleculeswith high accuracy. However, this mutant pore does not appear to havesensitivity for sequencing intact heteromeric ssDNA that passes throughthe pore.

Another exemplary biological pore is MspA, which has a funnel-like shapethat focuses the sensitivity of the ionic current to the bottom of thechannel. Moreover, achieving rate reduction of DNA through MspA andα-hemolysin can be achieved by using enzymes. As shown in FIG. 1 of(Manrao et al., Nature Biotechnology, 30:349-53, 2012), rate reductionof DNA through is achieved with the enzyme perched on the MspA nanopore.However, this results in non-deterministic sensing durations, repeatedreads induced by backtracking, and an inability to sense homopolymericregions. The mechanism of phi29 polymerase mediated DNA translocationwas developed in (Cherf et al., Nat. Biotech., 30(4):344-8, 2012) onα-hemolysin and implemented on the more sensitive MspA nanopore (Manraoet al., Nature Biotechnology, 30:349-53, 2012). Step-wise rates ofpolymerization-catalyzed translocation are 2.5-40 nt/s, meeting therequirements for DNA rate reduction. However, while enzymes on bioporescan reduce the rate of translocation, there is lack of control over thedwell time of each nucleotide, which will make blind tracking of repeatsvery difficult, and challenging to differentiate from long pauses on asingle nucleotide read. In terms of sensitivity, as shown in FIG. 3 of(Manrao et al., Nature Biotechnology, 30:349-2012), reading a repetitiveDNA template can be achieved with phi29 on MspA. The FIG. (3 a) shows anexample trace for a DNA template composed of repeated CATtrinucleotides, with the exception of one CAG triplet in the middle ofthe sequence. The * represent “toggles” detected (by human analysis) asrepeated transitions between two levels, which are intrinsic to theenzyme-based control method and incur read errors. The FIGS. 3 b) alsoshows Idealization of (3 a), with mean currents of levels aligned withthe known DNA sequence, and removing disparity of measured durationsshown in (3 a). Idealization shows a repeating pattern of three levelsinterrupted by the single dG substitution. Four levels are affected bythe single dG with the largest deviations closest to the substitution,suggesting residual current is principally influenced by ˜4 nucleotides.That the ionic current through MspA is influenced by ˜4 nucleotides mostproximal to the limiting aperture adds considerable complexity toidentifying the sequence. While one would ideally build a library ofdistinct current amplitudes that map to each of the 4⁴=256 combinations,as suggested in the art, such a library will be difficult to achieve.The reason is that identifying step transitions in channel currentrecordings requires a signal-to-noise ratio (SNR) of at least 2 withhalf-amplitude methods (SNR≧1.5 for Markov-based methods). With RMSnoise of 0.5 pA at recording bandwidths, amplitudes shifts must be atleast 1 pA to have the required SNR, resulting in only ˜40 detectablelevels within the amplitude range of 40 pA with MspA (or, at most 53levels at SNR 1.5). Moreover, fewer than 40 levels will be observedsince the range will not be uniformly utilized, and while furtherfiltering could reduce noise to add amplitude discrimination it alsoresults in missing more of the faster ssDNA motion transitions that arealready present.

Presently, there is no nanopore for which ionic current sensing canprovide single-nucleotide sensitivity for nucleic acid sequencing.Still, improvements to the sensitivity of biological pores andsolid-state pores (graphene) are active and ongoing research fields. Oneissue is that ionic current sensing does not permit direct tracking ofprogress through homolymeric regions (base repeats), since there is nodistinct signal-per-nucleotide of motion of homopolymeric ssDNA throughthe pore. Tracking repeats is essential, for example, since deletionsand insertions of specific mononucleotide repeats (7, 9 nt) within humanmitochondrial DNA have been implicated in several types of cancer(Sanchez-Cespedes, et al., Cancer Research, 61(19):7015-7019, 2001).While enzymes on biopores can reduce the rate of translocation, there islack of control over the dwell time of each nucleotide. On the otherhand, using a constant delivery rate with two-pore control,non-deterministic pauses are eliminated, and accurate estimation ofrepeat lengths can be made. Re-reading the repeat section many times canalso improve the estimation errors and identify error bounds, and thiscan be done without having to reverse the polymerization chemistrycaused by enzymes.

A recent study showed that, with a single nanopore, reduced rates cannotbe achieved by merely reducing the voltage (Lu et al., BiophysicalJournal, 101(1):70-9, 2011). Instead, as voltage is reduced, the ratesof a single-stranded DNA (ssDNA) become more random (includingbacktracking), since Brownian motion becomes an increasing contributorto velocity fluctuations. The study also shows that high voltage forceis required to suppress Brownian-motion induced velocity fluctuationsthat will otherwise confound sequencing measurements, even when using anidealized single-nucleotide-sensitive nanopore sensor.

The sequencing method provided in the present disclosure, based on atwo-pore device, provides a ready solution to these problems andadditional advantages over the existing methods. In concert with one ortwo pores that have sufficient sensitivity for sequencing, at high orlow voltage, the two pore control solves the sequencing rate controlproblem of single nanopore implementations. Such pores can includebiological pores housed in solid-state substrates, biological pores inmembranes formed across solid-state substrates, or solid-state pores(e.g., in graphene, silicon, or other substrates). In one aspect, anenzyme such as phi29 on a biological pore such as MspA can be used atone or both pores, with high voltages used to generate large signals forsequencing and a low differential voltage that generates a force on eachenzyme that is sufficient to hold the enzymes in position atop each poreand permit polymerization-catalyzed DNA motion, but not large enough tostall or dissociate the enzymes. Such a configuration can improve themethods in Cherf et al., Nat. Biotech., 30(4):344-8, 2012 and Manrao etal., Nature Biotechnology, 30:349-53, 2012, by significantly boostingthe measurement signal, and permitting two pores to read one stand ofDNA at the same time.

In addition, the method of the present disclosure can generatesufficiently high voltage at the pore to ensure detection sensitivity atthe pore using ionic current sensing. It is plausible that high voltagewould suppress Brownian motion enough to ensure constant rates througheach pore, and configuration of the DNA outside each pore will affectthe energetics of motion of DNA in either direction. Additionally, thevoltage competition used in the method (FIG. 1(III)) can be tuned sothat the molecule spends sufficient time in the pore to allow analysisof the molecule. Still further, the present method is free of the needfor enzymes, optics, or attachments to the DNA. Therefore, the methodprovides high signal-to-noise detection currents through the nanoporewhile regulating the molecule delivery rate, a capability that is notpossible with single nanopore implementations.

The method can be used to identify the composition of monomers in acharged polymer. In one aspect, the monomer unit is a nucleotide whenthe polymer is a single stranded DNA or RNA. In another aspect, themonomer unit can be a nucleotide pair, when the polymer is doublestranded.

In one aspect, the method can be used to identify a modification to thepolymer, such as a molecule bound to a monomer, in particular when thebound molecule is charged. The bound molecule does not have to becharged, however, as even a neutral molecule can change the ioniccurrent by virtue of its size.

In another aspect, the modification comprises the binding of a moleculeto the polymer. For instance, for a DNA molecule, the bound molecule canbe a DNA-binding protein, such as RecA, NF-κB and p53. In yet anotheraspect, the modification is a particle that binds to a particularmonomer or fragment. For instance, quantum dots or fluorescent labelsbound to a particular DNA site for the purpose of genotyping or DNAmapping can be detected by the device. Accordingly, the device of thepresent disclosure provides an inexpensive way for genotyping and DNAmapping as well, without limitation.

In one aspect, the polymer is attached to a solid support, such as abead, at one end of the polymer.

Also provided, in one embodiment, is a method for determining thesequence of a polynucleotide, comprising: (a) loading a samplecomprising a polynucleotide in the upper chamber of the device of any ofthe above embodiments, wherein the device is connected to a power supplyfor providing a first voltage between the upper chamber and the middlechamber, and a second voltage between the middle chamber and the lowerchamber, wherein the polynucleotide is optionally attached to a solidsupport at one end of the polynucleotide; (b) setting an initial firstvoltage and an initial second voltage so that the polynucleotide movesfrom the upper chamber to the middle chamber and from the middle chamberto the lower chamber, thereby locating the polymer across both the firstand second pores; (c) adjusting the first voltage and the second voltageso that both voltages generate force to pull the polynucleotide awayfrom the middle chamber, wherein the two voltages are different inmagnitude, under controlled conditions, so that the polynucleotide movesacross both pores in one direction and in a controlled manner; and (d)identifying each nucleotide of the polynucleotide that passes throughone of the pores, by measuring an ionic current across the pore when thenucleotide passes that pore.

EXAMPLES

The present technology is further defined by reference to the followingexamples. It will be apparent to those skilled in the art that manymodifications, both to threads and methods, may be practiced withoutdeparting from the scope of the current invention.

Example 1 Capture and Control of Individual dsDNA Molecules in Pores

This example shows that capture of DNA into each pore in a two-poredevice is readily detected as shift in each independent ionic porecurrent measured.

This example demonstrates dual-pore capture using dsDNA with and withouta bead attached to one end. Experiments with bead-tethered ssDNA canalso be explored.

Upon capture and stalling of the DNA, the pore voltage nearest the bead(V1, FIG. 1(I) in the case of capture from chamber A) can be reversedand increased until the competing force on the DNA draws it back towardchamber A. The ionic current in either pore can readily detect captureand exit of the DNA during the experiment.

When a bead is used, the bead has a proper size that prevents the beadfrom passing either or both of the pores. Methods that ensure a 1 to 1bead-DNA ratio have been developed in the art. For example, monovalentstreptavidin-coated Quantum dots (QDs; QD655, Invitrogen) conjugated tobiotinylated DNA duplexes (or ssDNA) can provide beads in the 20-30 nmdiameter range, with larger beads (30-100 nm) possible by using goldparticles or latex. The influence of bead on hydrodynamics and charge,as it relates to capture rate, can be considered in designing theexperiments.

Without beads, dsDNA passes through a pore at ˜0.1 ms/kbp. DNA oflengths 500 by and 4 kbp, and λ-phage dsDNA molecules (˜48 kbp) can beused. DNA samples can be delivered from chamber A into both pores, usinga common voltage polarity for each pore to promote capture from chamberA and passage through chamber B into chamber C (FIG. 1(I)). The largepersistence length of dsDNA (one Kuhn length is 100 nm) ensures that theDNA segment inside each pore is likely fully extended and rod-like.Voltage and ionic concentration can be varied to identify adequatecapture rates. Different buffered ionic concentrations can also be usedin each chamber to enhance or alter capture rates, and conductance shiftvalues that register the presence of DNA in each pore.

Using nanopore diameters 10 nm and larger minimizes the interaction(e.g., friction and sticking) between dsDNA and the nanopore walls. Forlarger pores, although dsDNA can be captured in an unfolded and foldedconfigurations, the single-file (unfolded) configuration is more likelyat higher voltages, and with shorter (≦3 kbp) dsDNA. For an inter-poredistance of 500 nm or less, it is contemplated that the probability ofdual-pore capture, following capture at the first pore (between chambersA and B) is very high, for voltages of at least 200 mV in 1 M KCl.

The radial distance within which voltage influence dominates thermaldiffusion, and leads to capture with high likelihood, has been estimatedto be at least 900 nm (larger than the inter-pore distance) for a rangeof pore sizes (6-15 nm diameter), voltages (120-500 mV), and with dsDNAat least 4 kbp in length (Gershow and Golovchenko, NatureNanotechnology, 2:775-779, 2007). These findings support a highlikelihood of prompt dual-pore capture of dsDNA, following single(first) pore capture of the dsDNA.

The capture and control of DNA through the two pores can benefit fromactive control hardware and real-time algorithms. The inventors havedeveloped active control hardware/software for DNA control. See, forexample, Gyarfas et al, Biophys. J., 100:1509-16, 2011); Wilson et al.,ACS Nano., 3(4):995-1003, 2009; and Benner et al., Nat. Nanotech.,2(11):718-24, 2007. A useful software is the LabVIEW software (Version8, National Instruments, Austin, Tex.), implemented on an FPGA(field-programmable gate array) system (PCI-7831R, NationalInstruments)). The referenced FPGA can control up to 4 amplifierssimultaneously. Further, the Axon Digidata 1440A Data Acquisition Systemused to digitize and log data onto a PC has 16 input channels, enough torecord voltage and current for up to 8 amplifiers in parallel. Otherreal-time operating system in concert with hardware/software forreal-time control and measurement could also be used for controlling theamplifiers, and digitizing and logging the data.

The inventors have also developed a low-noise voltage-clamp amplifiertermed the “Nanoclamp,” (Kim et al., IEEE Trans. On Biom. Circ. AndSyst. In press, May 2012; Kim et al., Elec. Lette., 47(15):844-6, July,2011; and Kim et al., Proceedings of the IEEE International SoC DesignConference (ISOCC), November, 2010) to functionalize and optimize theuse of one or more nanopores in small-footprint and multi-channeldevices. Any other commercial bench-top voltage-clamp or patch-clampamplifier, or integrated voltage-clamp or patch-clamp amplifier could beused for two pore control and measurement.

For a variety of solid-state pore materials and diameters, 0.1-10 kbptakes ˜1 ms to translocate. With a FPGA-controlled amplifier, one candetect capture and initiate competing voltage control within 0.020 ms,much faster than the 1 ms total passage time of 1 kbp DNA; thus,triggering the control method before DNA escapes (with no beadattachment) also has high likelihood. As demonstration of control, thetime to, and direction of, exit of the molecule from the pores can bedemonstrated as a function of the magnitude of and difference betweenthe competing voltages (FIG. 1(III)). In single pore experiments, largefluctuations in the velocity of long (≧1 kbp) dsDNA through the pore areexperimentally observed, and these fluctuations are too large to beattributed to diffusional Brownian motion. In (Lu, et al., BiophysicalJournal, 101(1):70-79, 2011), the dominant source of the net velocityfluctuations (i.e., DNA length divided by total passage time) wasmodeled as being due to viscous drag induced by the voltage affectingportions of the DNA not yet in the pore, in addition to portion in thepore, where the voltage region of attraction extends. The model matchedexperimental data reasonably well. Notably, if the center of mass of thedsDNA is colinear with the pore upon capture, net velocity is faster,but if it is offset from the pore, net velocity is slower. Whencompeting voltages are engaged in the two-pore device, dsDNA velocitieswill not be affected by this viscous-drag-induced perturbation, unlessthe voltage difference is sufficiently high. The reason is that, afterdsDNA capture through both pores and competing voltage is engaged, thedsDNA between the pores will be fully extended and rod-like, andtherefore cannot engage in creating structures near either interior poreentry. On the other hand, dsDNA structures on the exterior sides of thepores are constantly forced by each local pore voltage away from themiddle channel, and are thus less likely to confound the pore entrykinetics. Such structure may affect the controlled delivery kinetics,and calibration experiments can be used to quantify this.

Force uncertainty induced by random transverse DNA motion is likelyminimal. Additionally, the voltage force causes an electroosmotic flow(EOF) in the opposite direction of DNA motion, causing the DNA to moveslower than it would in the absence of the induced counterion flow.Since different radial positions of the molecule can give rise todifferent EOF fields in the nanopore, one issue is whether the effectivecharge density and therefore the net driving force vary enough duringfluctuations in DNA radial position to induce speed fluctuations. It isbelieved that the effective charge density of DNA in 1M KCl is stablefor a distance of 1 nm or more between the pore wall and the DNA.

Additionally, SiN nanopores have a negative surface charge thatintrinsically repels DNA. Thus, although the molecule will undergoradial position fluctuations, by using SiN pores with diameter greaterthan a few nanometers, it is likely that each constant voltage valuewill result in a constant effective force at each of the two pores, andthus a constant velocity in the direction of larger force when using twocompeting voltages in the two-pore setup. Treatment of other porematerial surfaces can produce comparable effects to that of SiN.

Velocity uncertainty induced by random translational DNA motion that iscaused by Brownian motion may be reduced by increasing the competingvoltages. Experiments can be carried out to determine whether suchreduction will occur. A single-nanopore study (Lu, et al., BiophysicalJournal, 101(1):70-79, 2011) supports that increasing the competingforces can reduce uncertainty caused by Brownian motion. The studyanalyzed the velocity fluctuations caused by Brownian motion, whichoccur on fast (nanosecond) time scales, and the sequencing errors thatresult from such fluctuations. Assuming a hypothetical and idealizedsingle-nucleotide sensor (noise-free detection at >22 MHz bandwidth),Brownian motion alone results in 75% read error. The relevant parameterfor predicting the error is k_(B)T/F*(0.34 nm), which is the ratio ofthermal energy to the work done to translocate the DNA the distance abetween nucleotides (0.34 nm). In the ratio, force F=Vλ is the voltage Vdriving DNA with charge density λ (0.2 e⁻/bp for dsDNA). For the presentcontrol method, increasing the voltage 50× results in 5% read error,with higher voltage further improving errors. With a single pore,however, since mean velocity v is F*d/(k_(B)T) with diffusion constantd, DNA speed also increases with F, placing even more unrealisticdemands on the sequencing bandwidth.

To maintain the 22 MHz bandwidth, a 50× increase in force with a singlenanopore would have to be paired with a 50× increase in solutionviscosity to maintain the same v. Practically, however, 22 MHz bandwidthis already much higher than any experimental nanopore platform hasdemonstrated, or promises to demonstrate, for single-nucleotidesequencing. Moreover, increasing viscosity can slow DNA only up to 10×(Fologea, et al., Nano Lett., 5(9):1734-7, 2005) with single nanopores.Using the two-pore platform, each voltage can be kept sufficiently high,and this may suppress fluctuations caused by Brownian motion, while thedifferential voltage that determines the net DNA speed can be adjustedto ensure control rates are within actual sequencing bandwidths(nominally 1 kHz). An alternative method of suppressing Brownian motioninduced velocity fluctuations is to use feedback control. In one aspect,with 10 kHz bandwidth of the second pore current feedback to actuationof the first pore voltage at 10 kHz bandwidth, Brownian motion can becompensated to control detectable features on the DNA to remain in andnear the second pore at these kHz closed-loop bandwidths. Thiscapability is a one-dimensional analogue to the anti-Brownianelectrokinetic (ABEL) trap that suppressed Brownian motion in twospatial dimensions and works by optical forcing of beads attached tomolecules at Hz closed-loop bandwidths (Wang and Moemer, ACS Nano,5:5792-9, 2011).

Example 2 Detection and Localization of RecA Filaments Bound to DNA

This example shows that the two-pore device can be used to map thebinding of a DNA-binding protein to dsDNA, and for proteins that have ordo not bind to specific sequences.

As demonstrated in Example 1, DNA samples can be captured from ChamberA. RecA protein catalyses an ATP-dependent DNA strand-exchange reactionthat pairs broken DNA with complementary regions of undamaged DNA. Usinga poorly hydrolyzable ATP analogue ATP γS, RecA filaments bound to dsDNAare very stable in high salt (e.g., 1M KCl) when first assembled inphysiological salt. As an alternative to ATPyS, which is slowlyhydrolyzed, this example can also use ADP-A1F4 (aluminum tetrafluoride),which does not turnover at all, and causes RecA to bind more tightly tothe DNA.

Detection of RecA filaments bound to λ-DNA through 20-30 nm nanoporeshas been demonstrated (Kowalczyk et al., Nano Lett., 10(1):324-8, 2010;Smeets et al., Nano Lett., 9(9):3089-95, 2009; and Hall et al. NanoLett., 9(12):4441-5, 2009], but filaments <20 by (6 or fewer RecAproteins) in length cannot be resolved using a single nanopore, due tothe coupling between translocation rate and measurement SNR.

Initial experiments of this example use bead-bound and unbound λ-DNAthat has been exposed to varying concentrations of RecA, to generate DNAthat is nearly uncoated, partially coated, and fully coated. Real-timemonitoring of each pore current can be used to gauge progress of thecontrolled delivery, and will be correlated for location mapping of thefilaments. Repeated measurements of each DNA will improve accuracy ofRecA mapping.

The added charge and bulk, and stability in high salt, when RecA isbound to DNA make it an ideal candidate to attempt detection andlocation mapping during controlled delivery with the proposedinstrument.

Control of RecA-bound DNA can also be attempted without a bead attachedto arrest translocation. As with dsDNA experiments in Example 1, activevoltage control can be used to promptly initiate competing voltagecontrol before the DNA exits the nanopores. As charged species that bindto DNA affect the mobility of DNA in an electric field, by altering thenet charge and stiffness of the DNA, motion control tuning experimentscan examine the influence of RecA binding to dsDNA on the force balanceused to control the motion of the dsDNA.

This example can demonstrate that the shortest observed filamentlengths, at low RecA concentrations, can be measured at high SNR and atsufficiently slow and controlled rates, so that any RecA protein boundin isolation can be detected if present.

The two-pore device therefore provides a completely new single-moleculeinstrument for basic research, as one could examine the capability todetect binding of additional proteins to the RecA-DNA filament, whichwould increase the filament width and thus be detected by a decrease inobserved current. For example, proteins that bind to the RecA-DNAfilament include LexA and bacteriophage lambda repressors, which useRecA to sense the status of the cell and switch on or off downstreamregulatory events.

Calibration experiments would involve detecting proteins that bind tospecific sequences (locations) on the DNA, so that protein-inducedshifts in the current would then permit estimation of the speed and ratecontrol performance of the DNA through the pores. Example proteins thatbind to specific sites on dsDNA include Lac repressor (binds to a 21 bpsegment), phage lambda repressor (which has multiple operator sites onλ-DNA), and other proteins.

Example 3 Detection and Localization of a Double-Stranded Segment withina Singled-Stranded DNA

This example demonstrates the production of up to 10 kb ssDNA withdoubled-stranded segments of varying lengths.

In a first step, 10 kbp dsDNA can be created by long range PCR. One endof the strand is biotinylated for bead attachment, and the strands areseparated by chemical denaturing. The unbeaded 10 kb ssDNA then servesas the measured strand in two-pore experiments. Complementarysingle-stranded segments with desired sizes can be created by PCRfollowed by bead capturing and strand separation.

ssDNA segments of varying lengths and at multiple sites within themeasured 10 kb ssDNA can be used, starting with a set of 100 ntsegments. Ionic current through a single solid-state pore was used todifferentiate dsDNA from ssDNA homopolymers, and purine and pyrimidinehomopolymers in (Skinner et al., Nano Lett., 9(8):2953-60, January2009). Thus, likelihood of differentiating single from double strandedsegments in DNA is high at sufficiently high voltage using the two-poredevice. Mapping ssDNA vs. dsDNA segments enables nanopore sequencingusing the hybridization-assisted method (though this method as ioproposed relies on a costly hybridization-assisted process), and can beused reveal both location and identity of target DNA sequences over longdistances (targeted sequencing). One can also explore the use of SingleStrand DNA Binding (SSB) proteins, as beads that will further amplifythe ssDNA vs. dsDNA differences in ionic current by binding to the ssDNAand creating a larger impedance than dsDNA.

Example 4 Capture and Control of Long ssDNA and Localization of RecA

This example demonstrates the capture and control of a long ssDNA andthe detection and localization of a RecA filament bound to the ssDNA.Additionally, it shows that the two-pore device can detect purine vs.pyrimidine homopolymeric segments within the ssDNA.

Stochastic detangling of 7 kb ssDNA through a 10 nm pore in a 20 nm SiNmembrane can be carried out as shown in Stefan et al., Nano Lett.,10:1414-20, 2010. While the single-nanopore method in Stefan et al. 2010unravels the ssDNA by the mechanical contact force between the tangledssDNA and the pore/membrane surface, it is contemplated that thedual-pore competing voltage setup can electrophoretically force ssDNA todetangle near and in between the pores at sufficiently high competingvoltages, by the action of each voltage force on the DNA backbonenearest each pore.

Detangling and subsequently precision control of the rate of ssDNAthrough the two pore setup is important for eventual sequencing of longssDNA molecules. At sufficiently high voltage (˜400 mV), it is possibleto discriminate purine and pyrimidine homopolymeric segments withinss-DNA (Skinner et al., Nano Lett., 9(8):2953-60, January 2009), whichis valuable for diagnostic applications and possibly cancer research.

This example also explores the use of RecA, or perhaps other SingleStrand DNA Binding (SSB) proteins, as detectable “speed-bumps” that aredifferentiable from the ssDNA ionic current by binding to the ssDNA andcreating a larger impedance. These speed bumps will allow directquantification of the controlled ssDNA speeds that are possible, whichin turn will demonstrate that the required 1 ms/nt is achievable. SinceRecA is not required to bind to specific trinucleotide sequence sites,but binds preferentially to TGG-repeating sequences and also tends tobind where RecA filaments are already formed, calibration experimentswill require the use of other ssDNA binding molecules that do bind tospecific known sequence locations. Having known binding sites that aredetectable as they pass through each pore is required to determine thespeed of the molecule as a function of the competing voltage values. Anon-limiting example is to use duplex strands (or bead-tethered duplexstrands) that hybridize to one or more known sites, from which theshifts in current could be used to detect passage of each duplex througheach pore, and then estimate the passing strand speed for the chosenvoltage values. Subsequently, RecA filaments can be formed and detectedon such molecules, keeping the duplex feature(s) as benchmark detectionpoints relative to which RecA filaments can be detected and theirposition inferred.

Methods for determining genetic haplotypes and DNA mapping byincorporating fluorescent labels into dsDNA (Xiao, et al., U.S. Pat. No.7,771,944 B2, 2010) can also use the two pore device, since the beadlabels (e.g., quantum dots, or any fluorescent label) is bulkier andwill produce shifts in the current just as binding proteins on dsDNAwould. Moreover, the two-pore method is simpler and much less expensivethan using high resolution imaging methods (i.e., total internalreflection fluorescence microscopy) to detect and map the labelpositions. It is also noted that any velocity fluctuations caused byBrownian motion during controlled delivery are much less deleterious fordetecting larger features (proteins, duplex segments, bead attachments)than for detecting smaller features.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

1. A method for controlling the movement of a charged polymer through apore, the method comprising the steps of: a) providing a dual-pore,dual-amplifier device for controlling the movement of a charged polymerthrough a first and second pore simultaneously, the device comprising(i) an upper chamber, a middle chamber and a lower chamber, (ii) a firstpore connecting the upper chamber and the middle chamber, and (iii) asecond pore connecting the middle chamber and the lower chamber; whereinthe device further comprises (iv) a power supply configured to provide afirst voltage between the upper chamber and the middle chamber, andprovide a second voltage between the middle chamber and the lowerchamber, each voltage being independently adjustable, and wherein thedevice provides (v) dual-amplifier electronics configured forindependent voltage control and current measurement at each pore, and(vi) wherein the first and second pores are configured so that thecharged polymer is capable of simultaneously moving across both pores ineither direction and in a controlled manner, b) loading a chargedpolymer in the upper chamber, middle chamber, or lower chamber of thedevice; and c) setting an initial first voltage and an initial secondvoltage so that at least a portion of the polymer moves through thefirst pore and the second pore; d) detecting the presence of the chargedpolymer in both pores simultaneously; and e) when the charged polymer isin both pores simultaneously, applying competing voltage to the firstand second pore to control the movement of the charged polymer throughboth pores simultaneously by adjusting the first voltage, the secondvoltage, or both.
 2. The method of claim 1, wherein applying competingvoltage control comprises reversing the direction of the first voltage,and adjusting the magnitude of the second voltage so that both voltagesnow generate force to pull the captured charged polymer away from themiddle chamber, towards the upper and lower chambers, and wherein saidcaptured charged polymer moves across both pores towards the upper orlower chamber in a controlled manner.
 3. The method of claim 2, whereinthe two voltages are different in magnitude.
 4. The method of claim 3,wherein the adjusted first voltage and second voltage at are 10 times to10,000 times as high, in magnitude, as the difference between the twovoltages.
 5. The method of claim 1, wherein the rate of translocation ofthe charged polymer through both nanopores simultaneously is 1ms/nucleotide or less.
 6. The method of claim 1, further comprisingidentifying a monomer unit of the io polymer by measuring an ioniccurrent across one of the pores when the monomer unit passes that pore.7. The method of claim 6, wherein the monomer unit is selected from thegroup consisting of: a nucleotide, a nucleotide pair, and an amino acidresidue.
 8. The method of claim 6, wherein the monomer unit is bound toa molecule.
 9. The method of claim 8, wherein the molecule is aDNA-binding protein.
 10. The method of claim 9, wherein the DNA-bindingprotein is a sequence-specific DNA-binding protein.
 11. The method ofclaim 9, wherein the DNA-binding protein is selected from the groupconsisting of: RecA, phage lambda repressor, NF-κB, and p53.
 12. Themethod of claim 1, wherein the charged polymer is a polypeptide.
 13. Themethod of claim 1, wherein the charged polymer is a polynucleotide. 14.The method of claim 13, wherein the polynucleotide is selected from thegroup consisting of a double-stranded DNA, single-stranded DNA,double-stranded RNA, single-stranded RNA, and DNA-RNA hybrid.
 15. Adual-pore, dual-amplifier device for controlling the movement of acharged polymer through a first and a second pore, the device comprising(i) an upper chamber, a middle chamber and a lower chamber, (ii) a firstpore connecting the upper chamber and the middle chamber, and (iii) asecond pore connecting the middle chamber and the lower chamber; whereinthe device further comprises (iv) a power supply configured to provide afirst voltage between the upper chamber and the middle chamber, andprovide a second voltage between the middle chamber and the lowerchamber, each voltage being independently adjustable, (v) wherein thedevice provides dual-amplifier electronics configured for independentvoltage control and current measurement at each pore, (vi) wherein thetwo voltages may be different in magnitude, and (vii) wherein the firstand second pores are configured so that the charged polymer is capableof simultaneously moving across both pores in either direction and in acontrolled manner.
 16. The device of claim 15, wherein the first poreand second pore are about 10 nm to 500 nm apart from each other.
 17. Thedevice of claim 15, wherein the first pore has a depth of at least about0.3 nm separating the upper chamber and the middle chamber and thesecond pore has a depth of at least about 0.3 nm separating the middlechamber and the lower chamber.
 18. The device of claim 15, wherein themiddle chamber is connected to a common ground relative to the twovoltages.
 19. A system comprising: a) a dual-pore, dual-amplifier devicefor controlling the movement of a charged polymer through a first andsecond pore, the device comprising (i) an upper chamber, a middlechamber and a lower chamber, (ii) a first pore connecting the upperchamber and the middle chamber, and (iii) a second pore connecting themiddle chamber and the lower chamber; wherein the device furthercomprises (iv) a power supply configured to provide a first voltagebetween the upper chamber and the middle chamber, and provide a secondvoltage between the middle chamber and the lower chamber, each voltagebeing independently adjustable, and (v) wherein the device providesdual-amplifier electronics configured for independent voltage controland current measurement at each pore, and (vi) wherein the two voltagesmay be different in magnitude, and (vii) wherein the first and secondpores are configured so that the charged polymer is capable ofsimultaneously moving across both pores in either direction and in acontrolled manner, and b) a charged polymer extending through both thefirst and second pores simultaneously, wherein the device is configuredto detect the charged molecule in both the first and second poressimultaneously.
 20. The system of claim 19, wherein the first voltageand second voltage are 10 times to 10,000 times as high, in magnitude,as the difference between the two voltages.
 21. The system of claim 19,wherein a net voltage differential between the first and second voltagesis present across the upper and lower chambers.
 22. The system of claim19, wherein the movement and the rate of movement of the charged polymeris controllable by the relative magnitude and direction of the first andsecond voltages.
 23. The system of claim 19, wherein the first pore andthe second pore are 0.3 nm to 100 nm in depth.